The enzyme soluble guanylate cyclase (sGC) is a heme-containing nucleotide cyclase that can be regulated by nitrovasodilators and free radicals such as nitric oxide. Soluble guanylate cyclase (sGC) is a cellular receptor for nitric oxide (NO) and takes part in transducing the NO signal to downstream effectors such as protein kinases, phosphodiesterases and ion channels. These downstream effectors mediate the physiological effects of NO. The regulation of physiological processes such as control of blood flow, platelet adhesion, endocrine function, neurotransmission, neuromodulation, and inflammation have been linked to activation of sGC by NO. Binding of NO to the heme of sGC triggers changes in the enzyme which activate its cyclization activity by 400-600-fold. Although it has been assumed that conformational changes in the enzyme mediate this activation, such changes have only recently been examined directly. Additionally, the in vivo activation of sGC by the heme-oxygenase product carbon monoxide (CO) has now been seen in vitro with the addition of the allosteric co-activator YC-1. In this proposal, the role of conformational changes in the activation of human soluble guanylate cyclase will be examined using a combination of CD, fluorescence, and mutagenesis techniques. The activation of the enzyme by NO, CO and YC-1 will be studied in an attempt to correlate structural changes in the enzyme with activation. FRET will be used to map the relative locations of several fluorophores and chromophores in the protein and the locations will be used as a monitor of how the conformation of the enzyme changes with activation. The binding site of the activator YC-1 will be studied by mutagenesis of residues proposed to be at the site. The mechanism of the inhibitor ODQ will be examined to show whether it binds to a site overlapping that of YC-1 and whether the inhibitor functions in ways other than heme-oxidation. It is hoped that through this work, a detailed mechanism of the activation can be determined, focussing on the structural changes in the enzyme.